PIH1D3-knockout rats exhibit full ciliopathy features and dysfunctional pre-assembly and loading of dynein arms in motile cilia.

Background: Recessive mutation of the X-linked gene, PIH1 domain-containing protein 3 (PIH1D3), causes familial ciliopathy. PIH1D3 deficiency is associated with the defects of dynein arms in cilia, but how PIH1D3 specifically affects the structure and function of dynein arms is not understood yet. To gain insights into the underlying mechanisms of the disease, it is crucial to create a reliable animal model. In humans, rats, and mice, one copy of the PIH1D3 gene is located on the X chromosome. Interestingly, mice have an additional, intronless copy of the Pih1d3 gene on chromosome 1. To develop an accurate disease model, it is best to manipulate the X-linked PIH1D3 gene, which contains essential regulatory sequences within the introns for precise gene expression. This study aimed to develop a tailored rat model for PIH1D3-associated ciliopathy with the ultimate goal of uncovering the intricate molecular mechanisms responsible for ciliary defects in the disease. Methods: Novel Pih1d3-knockout (KO) rats were created by using TALEN-mediated non-homologous DNA recombination within fertilized rat eggs and, subsequently, underwent a comprehensive characterization through a battery of behavioral and pathological assays. A series of biochemical and histological analyses were conducted to elucidate the identity of protein partners that interact with PIH1D3, thus shedding light on the intricate molecular mechanisms involved in this context. Results: PIH1D3-KO rats reproduced the cardinal features of ciliopathy including situs inversus, defects in spermatocyte survival and mucociliary clearance, and perinatal hydrocephalus. We revealed the novel function of PIH1D3 in cerebrospinal fluid circulation and elucidated the mechanism by which PIH1D3 deficiency caused communicating hydrocephalus. PIH1D3 interacted with the proteins required for the pre-assembly and uploading of outer (ODA) and inner dynein arms (IDA), regulating the integrity of dynein arm structure and function in cilia. Conclusion: PIH1D3-KO rats faithfully reproduced the cardinal features of ciliopathy associated with PIH1D3 deficiency. PIH1D3 interacted with the proteins responsible for the pre-assembly and uploading of dynein arms in cilia, and its deficiency led to dysfunctional cilia and, thus, to ciliopathy by affecting the pre-assembly and uploading of dynein arms. The resultant rat model is a valuable tool for the mechanistic study of PIH1D3-caused diseases.

Detergent-insoluble PFN1 inoculation expedites disease onset and progression in PFN1 transgenic rats.

Accumulating evidence suggests a gain of elusive toxicity in pathogenically mutated PFN1. The prominence of PFN1 aggregates as a pivotal pathological hallmark in PFN1 transgenic rats underscores the crucial involvement of protein aggregation in the initiation and progression of neurodegeneration. Detergent-insoluble materials were extracted from the spinal cords of paralyzed rats afflicted with ALS and were intramuscularly administered to asymptomatic recipient rats expressing mutant PFN1, resulting in an accelerated development of PFN1 inclusions and ALS-like phenotypes. This effect diminished when the extracts derived from wildtype PFN1 transgenic rats were employed, as detergent-insoluble PFN1 was detected exclusively in mutant PFN1 transgenic rats. Consequently, the factor influencing the progression of ALS pathology in recipient rats is likely associated with the presence of detergent-insoluble PFN1 within the extracted materials. Noteworthy is the absence of disease course modification upon administering detergent-insoluble extracts to rats that already displayed PFN1 inclusions, suggesting a seeding rather than augmenting role of such extracts in initiating neuropathological changes. Remarkably, pathogenic PFN1 exhibited an enhanced affinity for the molecular chaperone DNAJB6, leading to the sequestration of DNAJB6 within protein inclusions, thereby depleting its availability for cellular functions. These findings shed light on a novel mechanism that underscores the prion-like characteristics of pathogenic PFN1 in driving neurodegeneration in the context of PFN1-related ALS.

Characterization of Pore Size Distribution and Water Transport of UHPC Using Low-Field NMR and MIP.

Water transport is vital for the durability of ultra-high performance concrete (UHPC) in engineering, but its absorption behavior requires further comprehension. This study investigates the impact of silica fume (SF) and metakaolin (MK) on water absorption in UHPC matrix with a high volume of limestone powder (LS) under two curing temperatures, and the variation in water transport with pore size obtained by low field nuclear magnetic resonance (LF-NMR). Relations between cumulative water absorption with other properties were discussed, and the pore size distribution (PSD) measured by Mercury intrusion porosimetry (MIP) was compared with that determined by LF-NMR. Results showed that MK outperformed SF in reducing water absorption in UHPC matrix, containing 30% LS under steam curing due to the synergistic effect between MK and LS. The incorporation of LS greatly affected the water absorption process of UHPC matrix. In samples without LS, capillary and gel pores absorbed water rapidly within the first 6 h and slowly from 6 h to 48 h simultaneously. However, in samples with 30% LS, gel pore water decreased during water absorption process due to the coarsening of gel pores. MK was able to suppress gel pore deterioration caused by the addition of a large amount of LS. Compared with PSD measured by MIP, NMR performed better in detecting micropores (<10 nm).

Detergent-insoluble inclusion constitutes the first pathology in PFN1 transgenic rats.

Mutation of profilin 1 (PFN1) can cause amyotrophic lateral sclerosis (ALS). To assess how PFN1 mutation causes the disease, we created transgenic rats with human genomic DNA that harbors both the coding and the regulatory sequences of the human PFN1 gene. Selected transgenic lines expressed human PFN1 with or without the pathogenic mutation C71G at a moderate and a comparable level and in the similar pattern of spatial and temporal expression to rat endogenous PFN1. The artificial effects of arbitrary transgene expression commonly observed in cDNA transgenic animals were minimized in PFN1 transgenic rats. Expression of the mutant, but not the wild type, human PFN1 in rats recapitulated the cardinal features of ALS including the progressive loss of motor neurons and the subsequent denervation atrophy of skeletal muscles. Detergent-insoluble PFN1 inclusions were detected as the first pathology in otherwise asymptomatic transgenic rats expressing mutant human PFN1. The findings suggest that protein aggregation is involved in the neurodegeneration of ALS associated with PFN1 mutation. The resulting rat model is useful to mechanistic study on the ALS.

Diagnosis of Clostridium difficile infection using an UPLC-MS based metabolomics method.

INTRODUCTION: The fecal metabolome of Clostridium difficile (CD) infection is far from being understood, particularly its non-volatile organic compounds. The drawbacks of current tests used to diagnose CD infection hinder their application. OBJECTIVE: The aims of this study were to find new characteristic fecal metabolites of CD infection and develop a metabolomics model for the diagnosis of CD infection. METHODS: Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) was used to characterize the fecal metabolome of CD positive and negative diarrhea and healthy control stool samples. RESULTS: Diarrhea and healthy control samples showed distinct clusters in the principal components analysis score plot, and CD positive group and CD negative group demonstrated clearer separation in a partial least squares discriminate analysis model. The relative abundance of sphingosine, chenodeoxycholic acid, phenylalanine, lysophosphatidylcholine (C16:0), and propylene glycol stearate was higher, and the relative abundance of fatty amide, glycochenodeoxycholic acid, tyrosine, linoleyl carnitine, and sphingomyelin was lower in CD positive diarrhea groups, than in the CD negative group. A linear discriminant analysis model based on capsiamide, dihydrosphingosine, and glycochenodeoxycholic acid was further constructed to identify CD infection in diarrhea. The leave-one-out cross-validation accuracy and area under receiver operating characteristic curve for the training set/external validation set were 90.00/78.57%, and 0.900/0.7917 respectively. CONCLUSIONS: Compared with other hospital-onset diarrhea, CD diarrhea has distinct fecal metabolome characteristics. Our UPLC-MS metabolomics model might be useful tool for diagnosing CD diarrhea.

Risk factors and medical costs for healthcare-associated carbapenem-resistant Escherichia coli infection among hospitalized patients in a Chinese teaching hospital.

BACKGROUND: The emergence and spread of Carbapenem-resistant Escherichia coli (CREC) is becoming a serious problem in Chinese hospitals, however, the data on this is scarce. Therefore, we investigate the risk factors for healthcare-associated CREC infection and study the incidence, antibiotic resistance and medical costs of CREC infections in our hospital. METHODS: We conducted a retrospective, matched case-control-control, parallel study in a tertiary teaching hospital. Patients admitted between January 2012 and December 2015 were included in this study. For patients with healthcare-associated CREC infection, two matched subject groups were created; one group with healthcare-associated CSEC infection and the other group without infection. RESULTS: Multivariate conditional logistic regression analysis demonstrated that prior hospital stay (<6 months) (OR:3.96; 95%CI:1.26-12.42), tracheostomy (OR:2.24; 95%CI: 1.14-4.38), central venous catheter insertion (OR: 8.15; 95%CI: 2.31-28.72), carbapenem exposure (OR: 12.02; 95%CI: 1.52-95.4), urinary system disease (OR: 16.69; 95%CI: 3.01-89.76), low hemoglobin (OR: 2.83; 95%CI: 1.46-5.50), and high blood glucose are associated (OR: 7.01; 95%CI: 1.89-26.02) with CREC infection. Total costs (p = 0.00), medical examination costs (p = 0.00), medical test costs (p = 0.00), total drug costs (p = 0.00) and ant-infective drug costs (p = 0.00) for the CREC group were significantly higher than those for the no infection group. Medical examination costs (p = 0.03), total drug costs (p = 0.03), and anti-infective drug costs (p = 0.01) for the CREC group were significantly higher than for the CSEC group. Mortality in CREC group was significantly higher than the CSEC group (p = 0.01) and no infection group (p = 0.01). CONCLUSION: Many factors were discovered for acquisition of healthcare-associated CREC infection. CREC isolates were resistant to most antibiotics, and had some association with high financial burden and increased mortality.

Changes in antimicrobial use prevalence in China: results from five point prevalence studies.

OBJECTIVE: The abuse of antimicrobials is a serious concern in China. Several measures have been taken to improve the rational use of antimicrobials, including the establishment of a national surveillance network for antimicrobial use. This study describes the dynamic changes in antimicrobial use in China between 2001 and 2010, with the scope of identifying targets to improve the prescription of antimicrobials. METHODS: Five point prevalence surveys were performed in hospitals across mainland China in 2001, 2003, 2005, 2008, and 2010. All inpatients who were admitted for at least 24 hours were included in the study. Details regarding antimicrobial use by these patients and the collection of samples for bacterial culture from inpatients administered therapeutic antimicrobials were recorded. RESULTS: The surveys encompassed tertiary hospitals from all 31 provinces of mainland China. Antimicrobial use prevalence decreased from 54.79% in 2001 to 46.63% in 2010. While this decline was observed in most hospital departments, antimicrobial use remained stable or increased in others. Antimicrobial use prevalence was relatively high in the Pediatrics departments and general intensive care units, whereas it was lower in the Obstetrics (Neonatal group) departments in each survey. The proportion of patients administered a single antimicrobial increased from 60.78% in 2001 to 70.16% in 2010, while the proportion of administration of two or more antimicrobials declined. The bacterial culture rate increased from 25.22% in 2003 to 34.71% in 2010. Antimicrobial use prevalence (47.96% vs 46.16%), bacterial culture rate (36.40% vs 34.19%), and the proportion of administration of a single antimicrobial (71.41% vs 67.33%) were higher in teaching hospitals than in nonteaching hospitals in 2010. CONCLUSION: Although measures for enhancing the rational use of antimicrobials have been effective, further improvements are required. The findings from this study can promote such improvements.